More specifically, this invention relates to the biosensors that are used to measure the amount of analytes in bodily fluids. Optical methods are often used for making such measurements, but the present invention relates to improvements in electrochemical biosensors. While the method to be described herein can be applied to measurement of other analytes, including cholesterol, urea, creatinine, and creatine, measuring glucose in whole blood is of particular interest. Although the description here will emphasize the inventions application to measuring glucose, it should be understood that the invention has broader applications.
The invention relates to an electrochemical instrument in which a potential is applied to electrodes in contact with a biological sample and reagents. The resulting current is measured while the analyte is reacting with the reagents, and then correlated with the amount of an analyte in the sample. Such instruments are referred to as amperometric, in contrast with coulometric instruments that measure the total charge in coulombs produced from reaction of the sample by integrating the current over the entire measurement time. The amperometric instruments have an advantage in that they are less volume and time dependent. They do not wait for the entire volume of the analyte to be reacted, but only take measurements of the analyte by sampling the reaction rate at a predetermined time.
Many designs for such biosensors have been described in the art, for example, published U.S. Patent Application 2001/0042683. The electrodes are generally described as the working electrode and as the counter electrode. The electrodes are in contact with a solid layer containing reagents that oxidize the analyte in the sample, such as glucose oxidase, and mediators that reoxidize the reduced enzyme. The reduced mediator itself is oxidized at the working electrode, which produces a measurable current. This current is used to calculate the amount of glucose in the sample being tested, since it is an indirect measure of the oxidation of glucose in the sample. The reactions may be described by the following steps:
Glucose+Eoxid→Ered+Product (Gluconic Acid, Gluconolactone)
Ered+Medoxid→Medred+Eoxid 
Medred→Medoxid+n e−
Where Eoxid and Ered are oxidized and reduced forms of the redox center of the enzyme and Medoxid and Medred are the oxidized and reduced forms of the mediator.
For measuring glucose, the enzyme may be glucose oxidase and the mediator ferricyanide. Measuring other analytes will employ suitable enzymes and mediators. Typical combinations of enzyme, mediator and analyte are listed in Table 1.
TABLE 1Selected Substrates, Enzyme and Mediator SystemsAnalyteEnzymeMediatorGlucoseGlucose OxidaseFerricyanideGlucoseGlucose DehydrogenaseFerricyanideCholesterolCholesterol OxidaseFerricyanideLactateLactate OxidaseFerricyanideUric AcidUricaseFerricyanideAlcoholAlcohol OxidasePhenylenediamine
In order to assure accurate measurements, control solutions containing known amounts of glucose are used to verify that the instrument is operating properly. The composition of control solutions has been the subject of a number of patents and publications. Representative are U.S. Pat. Nos. 3,920,580; 4,572,899; 4,729,959; 5,028,542 and 5,605,837; WO 93/21928; WO 95/13535; and WO 95/13536. While control solutions containing blood serum have been used, more recent patents have been concerned with replacing serum-based control solutions with solutions free of serum, since serum-free solutions are more consistent and stable than those containing serum. The control solution should contain a known concentration of glucose in a serum-like matrix to determine the accuracy of both the enzymatic biosensor and the potentiostat meter. It will be evident that the composition must be stable over lengthy periods of storage before use.
Control solutions should serve the purpose of checking the glucose monitoring system's functioning, but at the same time they should be identified and separated from the readings of real blood samples. This is because the control solutions contain known amounts of glucose and provide readings that have no therapeutic purpose. If the control solutions cannot be identified and their responses separated from those of the blood samples by the test meter, glucose readings of the control solutions will be included in the history of the glucose measurements, which could lead to wrong interpretation of a patient's diabetic condition. Or, if a control solution is substituted for a blood sample, it may be mistakenly considered by a physician as indicating a need to change treatment. Furthermore, since the temperature response of the control solutions is different from that of the blood samples, temperature compensation for measurements made at temperatures other than 25° C. will be less accurate if a test meter cannot distinguish between blood samples and control solutions. Therefore, it is highly desirable that the glucose monitoring system automatically detect and identify the control solutions in order to separate the glucose readings of control solutions from those of the blood samples, and to provide separate temperature compensation to both the blood samples and the control solutions.
There have been several patents describing methods of identifying the control solutions through various mechanisms. In U.S. Pat. No. 5,723,284, electrochemical measurement of glucose in blood is discussed. The '284 patent proposed to modify the control solutions, changing the ratio of current readings taken from two oxidation periods separated by a rest period. The meter would recognize that a control solution was being measured and take appropriate action to prevent the results from being included in the blood sample results. The '284 patent also teaches that the control solution should be buffered in a pH range of 4.8 to 7.5 to be effective.
Another method for determining whether a control solution or a blood sample is being measured for its glucose content is disclosed in U.S. Published Application 2002/0139692A1. An index is determined that relates the decline of electrical current to the nature of the sample being tested.
U.S. Pat. Nos. 5,620,579 and 5,653,863, proposed to begin the test of a sample by providing an initial positive potential pulse for a short period in order to reoxidize any prematurely reduced mediator. Such an initial pulse was referred to as a “burnoff period”.
When a potential is applied across the working and counter electrodes and a liquid sample is introduced to the sensor, the dry reagents are rehydrated by the liquid sample and current begins to flow, typically increasing to a peak and then declining over the “burn period,” usually about ten seconds in length. During this period the previously reduced mediator is reoxidized to reduce the bias towards incorrect high results. If a full amount of sample is not present, additional error may be introduced since all of the reagents may not become available for reaction or the working and counter electrodes might not be in complete contact with sample, thus reducing the current during the “burn” period.
After the burn period has been completed, a rest period is provided at a lower potential or at no potential (open circuit). During this rest period the glucose oxidation reaction continues to take place and the mediator is reduced. Then, a constant potential is applied again between the working and counter electrodes and the current is measured for a short period, typically about two to ten seconds. The current is initially high, but it declines rapidly as diffusion of the mediator begins to control. At a predetermined time, the measured current is used to determine the glucose content of the sample.
Adding an internal reference compound is a common practice in analytical chemistry to provide a quantitative reference signal. This working principle has been used in a recent published patent application No. WO 2005/078118, where an internal reference is added to the reagent system to achieve some formulation purpose.
In WO2004/040286A1, it is proposed that the control solution include a reducing substance chosen from uric acid, bilirubin, ascorbic acid, methylene blue, Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid, and acetaminophen, thus changing the ratio of current readings taken from two oxidation periods separated by a rest period and enabling the control solution to be identified.
The present inventors have sought an improved method of distinguishing control solutions from biological samples. Their methods are described in detail below.